Hydrogen production by the unicellular, diazotrophic cyanobacterium Cyanothece sp. strain ATCC 51142 under conditions of continuous light

Abstract

We report on the hydrogen production properties of the unicellular, diazotrophic cyanobacterium Cyanothece sp. strain ATCC 51142. This organism has a versatile metabolism and can grow in the presence or absence of combined nitrogen and can grow photosynthetically or mixotrophically and heterotrophically in the presence of glycerol. The strain produces a bidirectional hydrogenase (encoded by the hox genes), an uptake hydrogenase (hupLS), and nitrogenase (nifHDK). We demonstrated hydrogen production by both the hydrogenase and the nitrogenase under appropriate metabolic conditions. The highest rates of hydrogen production were produced under nitrogen-fixing conditions when cells were grown and incubated under continuous light conditions, in either the presence or absence of glycerol. Under such nitrogen-fixing conditions, we have achieved rates of 300 micromol H(2)/mg chloramphenicol (Chl)/hr during the first 24 h of incubation. The levels of H(2) measured were dependent upon the incubation conditions, such as sparging with argon, which generated anaerobic conditions. We demonstrated that the same conditions led to high levels of H(2) production and N(2) fixation, indicating that low-oxygen conditions favor nitrogenase activity for both processes. The levels of hydrogen produced by the hydrogenase are much lower, typically 5 to 10 micromol H(2)/mg Chl/hr. Hydrogenase activity was dependent upon electron transport through photosystem II (PS II), whereas nitrogenase activity was more dependent on PS I, as well as on respiration. Although cells do not double under the incubation conditions when sparged with argon to provide a low-oxygen environment, the cells are metabolically active, and hydrogen production can be inhibited by the addition of chloramphenicol to inhibit protein synthesis.

FIG. 1.

H₂ production by cultures of Cyanothece sp. strain ATCC 51142 grown in ASP2 without combined nitrogen (ASP2−) after subculture from a culture grown for 14 days in ASP2 medium. □, sparged with argon; ⋄, sparged with air.

FIG. 2.

H₂ production by cultures of Cyanothece sp. strain ATCC 51142 grown in ASP2 without combined nitrogen (ASP2−) with 50 mM glycerol and incubated under different conditions, with and without sparging with argon (Arg) and 20 μg/ml chloramphenicol (Cm), as indicated. Nitrogenase is responsible for high levels of hydrogen production in ASP2− medium under anaerobic conditions with glycerol, and de novo protein synthesis is needed.

FIG. 3.

(a) H₂ production by cultures of Cyanothece sp. strain ATCC 51142 grown in ASP2 medium without combined nitrogen (ASP2−) with 50 mM glycerol, incubated under either the LL (□) or DD (▪) condition, and sparged with argon. (b) H₂ production by cultures of Cyanothece sp. strain ATCC 51142 grown in ASP2− medium with 50 mM glycerol, incubated under either the LL (▵) or DD (▴) condition, and sparged with air.

FIG. 4.

H₂ production by cultures of Cyanothece sp. strain ATCC 51142 grown in either regular ASP2 medium without combined nitrogen (ASP2−; pH 7.9) (○) or in ASP2− medium modified (with one-fifth the amount of Ca²⁺ and 10-fold Fe³⁺) at pH 7.4 (□) or pH 8.4 (•). All samples were sparged with argon.